Cancer research progress, having benefited greatly from studies on powerful model systems, supports the hypothesis that specific mutations leading to decreased genome stability are critical early events in tumorigenesis. Experiments in yeast and mice show that eukaryotic genome integrity requires the action of the multifunctional enzyme Flap EndoNuclease (FEN-1), and that mutations in FEN-1 result in DNA duplication defects that occur in human tumors and inherited human diseases. Working together with collaborators, the Tainer lab has discovered features of FEN-1, Rnase HII and the processivity factor for DNA polymerase, termed proliferating cell nuclear antigen (PCNA) that support molecular mechanisms for the activities, regulation and coordination of these proteins and of their dynamic complexes in DNA replication and repair. Our experimentally derived hypothesis, which we call the "rotary hand-off" model, encompasses mechanisms for DNA recognition, precise cleavage to yield ligatable DNA ends, reduced mutations by preventing the generation of gapped or flapped templates, and coordination rather than interference by key proteins that must associate with PCNA. In this renewal, we propose to test individual features of the rotary hand-off model with respect to specificity, activity, and coordination of repair and replication events by PCNA:FEN-1 and related complexes. In concert, the proposed experiments on archaeal, yeast, and human proteins aim to provide the biophysical, biochemical and biological results for a unified understanding of the role of these proteins in genetic integrity and cancer resistance. Overall, this research aims to provide fundamental knowledge on FEN-1 and related complexes relevant to defining their roles in the regulation of genome fidelity and the mechanisms whereby loss of the functions of these coordinated complexes may lead to inheritable genetic defects and the initiation of cancer.